Eosinophils and Bacteria, the Beginning of a Story.

Eosinophils are granulocytes primarily associated with TH2 responses to parasites or immune hyper-reactive states, such as asthma, allergies, or eosinophilic esophagitis. However, it does not make sense from an evolutionary standpoint to maintain a cell type that is only specific for parasitic infections and that otherwise is somehow harmful to the host. In recent years, there has been a shift in the perception of these cells. Eosinophils have recently been recognized as regulators of immune homeostasis and suppressors of over-reactive pro-inflammatory responses by secreting specific molecules that dampen the immune response. Their role during parasitic infections has been well investigated, and their versatility during immune responses to helminths includes antigen presentation as well as modulation of T cell responses. Although it is known that eosinophils can present antigens during viral infections, there are still many mechanistic aspects of the involvement of eosinophils during viral infections that remain to be elucidated. However, are eosinophils able to respond to bacterial infections? Recent literature indicates that Helicobacter pylori triggers TH2 responses mediated by eosinophils; this promotes anti-inflammatory responses that might be involved in the long-term persistent infection caused by this pathogen. Apparently and on the contrary, in the respiratory tract, eosinophils promote TH17 pro-inflammatory responses during Bordetella bronchiseptica infection, and they are, in fact, critical for early clearance of bacteria from the respiratory tract. However, eosinophils are also intertwined with microbiota, and up to now, it is not clear if microbiota regulates eosinophils or vice versa, or how this connection influences immune responses. In this review, we highlight the current knowledge of eosinophils as regulators of pro and anti-inflammatory responses in the context of both infection and naïve conditions. We propose questions and future directions that might open novel research avenues in the future.

Genomic plasticity and antibody response of Bordetella bronchiseptica strain HT200, a natural variant from a thermal spring.

Classical Bordetella species are primarily isolated from animals and humans causing asymptomatic infection to lethal pneumonia. However, isolation of these bacteria from any extra-host environmental niche has not been reported so far. Here, we have characterized the genomic plasticity and antibody response of Bordetella bronchiseptica strain HT200, isolated from a thermal spring. Genomic ANI value and SNPs-based phylogenetic tree suggest a divergent evolution of strain HT200 from a human-adapted lineage of B. bronchiseptica. Growth and survivability assay showed strain HT200 retained viability for more than 5 weeks in the filter-sterilized spring water. In addition, genes or loci encoding the Bordetella virulence factors such as DNT, ACT and LPS O-antigen were absent in strain HT200, while genes encoding other virulence factors were highly divergent. Phenotypically, strain HT200 was non-hemolytic and showed weak hemagglutination activity, but was able to colonize in the respiratory organs of mice. Further, both infection and vaccination with strain HT200 induced protective antibody response in mouse against challenge infection with virulent B. bronchiseptica strain RB50. In addition, genome of strain HT200 (DSM 26023) showed presence of accessory genes and operons encoding predicted metabolic functions pertinent to the ecological conditions of the thermal spring.

Efficacy of Rg1-Oil Adjuvant on Inducing Immune Responses against Bordetella bronchiseptica in Rabbits.

Bordetella bronchiseptica (B. bronchiseptica) is an obligately aerobic, oxidase- and catalase-positive, nonfermentative Gram-negative coccobacillus. This study is aimed at examining the immune effects of Rg1, Rg1 plus oil, and other common adjuvants on inactivated B. bronchiseptica vaccine in rabbits. The mechanism underlying the adjuvant effect of Rg1 plus oil on the vaccine was also explored. Rg1 (100 µg) plus oil significantly improved the immune effect of B. bronchiseptica vaccine at both the humoral and cellular levels. Rg1-oil adjuvant increased the levels of IL-2 and IL-4 in rabbits after immunization. Rg1 (100 µg) plus oil also significantly increased TLR2 expression and downregulated NF-κB in splenocytes. Rg1-oil adjuvant may increase the levels of IL-2 and IL-4 via upregulating TLR2, thereby enhancing the immune effect of B. bronchiseptica vaccine. In conclusion, Rg1 plus oil could be used as a potential vaccine adjuvant for rabbit B. bronchiseptica vaccine.

Does Bordetella pertussis vaccine offer any cross-protection against Bordetella bronchiseptica? Implications for pet owners with cystic fibrosis.

WHAT IS KNOWN AND OBJECTIVE: The Gram-negative bacterium, Bordetella bronchiseptica, causes lower airway respiratory disease in people with cystic fibrosis (CF), as well as in companion animals, especially dogs. Presently, there are several acellular vaccines available for B. pertussis but no vaccine available for B. bronchiseptica. However given the shared protein homology between these two closely related species, we wished to explore whether pertussis vaccines may offer some cross-protection against B. bronchiseptica. COMMENT: Bordetella pertussis and B. bronchiseptica are closely related phylogenetically, as well as sharing protein homology in several pertussis vaccine components, including (i) pertussis toxin (PT), (ii) filamentous haemagglutinin (FHA), (iii) pertactin and (iv) fimbriae (types 2 and 3). Given that pertussis vaccine contains cross-reactive antigens with B. bronchiseptica, licensed pertussis vaccines may therefore offer cross-protection against B. bronchiseptica. WHAT IS NEW AND CONCLUSION: Cystic fibrosis pet owners should ensure that they have an up-to-date vaccination record relating to their pertussis vaccine. Although no monovalent human pertussis vaccines are currently available, licensed non-live booster vaccines for B. pertussis are available for individuals in the age range >10 years old. People with CF should ensure that they are adequately and currently protected against pertussis, to avoid whooping cough, which may also offer some cross-protection against B. bronchiseptica and therefore help further mitigate the risk of zoonotic infection of this organism from pets to their owners.

PEGylated nano-Rehmannia glutinosa polysaccharide induces potent adaptive immunity against Bordetella bronchiseptica.

Vaccines, in many cases, stimulate only too weak immunogenicity to prevent infection. Therefore, adjuvants are required during their preparation to boost the immune response. We herein developed a PEGylated nano-adjuvant based on Rehmannia glutinosa polysaccharide (RGP). The addition of PEG layer exhibits enhanced immune performance of the nano-RGP. Stimulation of dendritic cells (DCs) with PEGylated nano-RGP (pRL) led to increased proliferation and cytokine production (IL-6, IL-12, IL-1ß and TNF-α). The pRL was internalized into DCs via a rapid and efficient method. The mice immunized with pRL exhibited enhanced antigen-specific serum IgG and Th1-(IFN-γ), Th2-(IL-4), and Th17-(IL-17, IL-6) cytokine production, contributing to a good anti-infection performance. Furthermore, the pRL could effectively deliver the antigen to the lymph nodes (LNs), activate DC in the LN and produce enhanced CD4+and CD8+ T-cells-derived memory (CD44high CD62Lhigh), and effector (CD44high CD62Llow) as well as functional phenotypes. Our results revealed that pRL can act as a promising adjuvant with targeted delivery of antigen due to its effective activation and robust adaptive immunity induction of DCs.

Specific Integration of Temperate Phage Decreases the Pathogenicity of Host Bacteria.

Temperate phages are considered as natural vectors for gene transmission among bacteria due to the ability to integrate their genomes into a host chromosome, therefore, affect the fitness and phenotype of host bacteria. Many virulence genes of pathogenic bacteria were identified in temperate phage genomes, supporting the concept that temperate phages play important roles in increasing the bacterial pathogenicity through delivery of the virulence genes. However, little is known about the roles of temperate phages in attenuation of bacterial virulence. Here, we report a novel Bordetella bronchiseptica temperate phage, vB_BbrS_PHB09 (PHB09), which has a 42,129-bp dsDNA genome with a G+C content of 62.8%. Phylogenetic analysis based on large terminase subunit indicated that phage PHB09 represented a new member of the family Siphoviridae. The genome of PHB09 contains genes encoding lysogen-associated proteins, including integrase and cI protein. The integration site of PHB09 is specifically located within a pilin gene of B. bronchiseptica. Importantly, we found that the integration of phage PHB09 significantly decreased the virulence of parental strain B. bronchiseptica Bb01 in mice, most likely through disruption the expression of pilin gene. Moreover, a single shot of the prophage bearing B. bronchiseptica strain completely protected mice against lethal challenge with wild-type virulent B. bronchiseptica, indicating the vaccine potential of lysogenized strain. Our findings not only indicate the complicated roles of temperate phages in bacterial virulence other than simple delivery of virulent genes but also provide a potential strategy for developing bacterial vaccines.

Immunogenicity of recombinant outer membrane porin protein and protective efficacy against lethal challenge with Bordetella bronchiseptica in rabbits.

AIMS: The outer membrane porin protein (OMPP) of Bordetella bronchiseptica is an important adhesion factor and protective immunogen. The aim of this study was to verify the immunogenicity of recombinant OMPP and its protective efficacy against a lethal challenge with B. bronchiseptica in rabbits. METHODS AND RESULTS: Soluble rOMPP was successfully expressed in Escherichia coli, and the purified recombinant protein was mixed with the ISA 201 VG adjuvant to prepare a subunit vaccine for B. bronchiseptica. Rabbits were immunized with the rOMPP subunit vaccine and then infected with the virulent B. bronchiseptica strain QDBb01. Rabbits immunized with the subunit vaccine were completely protected compared to the control group, and the protective effect was obviously better than that of the inactivated whole-cell vaccine. Moreover, analysis of the immunization duration showed that the rOMPP subunit vaccine provided immune protection for at least 4 months after the second immunization. CONCLUSIONS: The rOMPP subunit vaccine completely protected rabbits from a subsequent B. bronchiseptica challenge. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will provide key information for the development of a safe and effective recombinant subunit vaccine against B. bronchiseptica in rabbits.

Subcutaneous Immunization of Dogs With Bordetella bronchiseptica Bacterial Ghost Vaccine.

The Bordetella species are Gram-negative bacterial pathogens that colonizes mammalian respiratory tract causing respiratory diseases in humans and animals. B. bronchiseptica causes clinical conditions in many mammals including immunocompromised humans. Using the dog model of respiratory infection, it has been shown in this study that a newly developed B. bronchiseptica Bacterial Ghost (BbBG) vaccine exhibited significant protection in the face of a severe pathogenic bacterial challenge in seronegative dogs. The protein E-specific lysis mechanism was used to produce BbBGs. Bacterial Ghosts (BGs) are the empty cell envelope of Gram-negative bacterium. They are genetically processed to form a microscopic hole in their membrane, through which all the cytoplasmic contents are expelled leaving behind intact empty bacterial shells. Due to the intact surface structures of BGs, they offer the safety of inactivated but efficacy of live attenuated vaccines. In this study, seronegative dogs were vaccinated subcutaneously (s/c) with two different doses of a newly developed BbBG vaccine [lower 10∧5 (BbBG - 5) and higher 10∧7 (BbBG - 7)] on day 0 and 21. The animals were challenged (by aerosol) with virulent live B. bronchiseptica strains 41 days after first vaccination. The dogs vaccinated s/c with BbBG - 7 vaccine had significantly lower spontaneous coughing scores (P = 0.0001) than dogs in negative control group. Furthermore, the tested BbBG - 7 vaccine was equivalent to the positive control vaccine Bronchicine CAe in terms of safety and efficacy. For the first time, we report the successful use of liquid formulated BGs vaccines in animal studies. Earlier reported studies using BGs vaccines were performed with resuspended freeze-dried BGs preparations.

Bordetella Colonization Factor A (BcfA) Elicits Protective Immunity against Bordetella bronchiseptica in the Absence of an Additional Adjuvant.

Bordetella bronchiseptica is an etiologic agent of respiratory diseases in animals and humans. Despite the widespread use of veterinary B. bronchiseptica vaccines, there is limited information on their composition and relative efficacy and on the immune responses that they elicit. Furthermore, human B. bronchiseptica vaccines are not available. We leveraged the dual antigenic and adjuvant functions of Bordetella colonization factor A (BcfA) to develop acellular B. bronchiseptica vaccines in the absence of an additional adjuvant. BALB/c mice immunized with BcfA alone or a trivalent vaccine containing BcfA and the Bordetella antigens FHA and Prn were equally protected against challenge with a prototype B. bronchiseptica strain. The trivalent vaccine protected mice significantly better than the canine vaccine Bronchicine and provided protection against a B. bronchiseptica strain isolated from a dog with kennel cough. Th1/17-polarized immune responses correlate with long-lasting protection against bordetellae and other respiratory pathogens. Notably, BcfA strongly attenuated the Th2 responses elicited by FHA and Prn, resulting in Th1/17-skewed responses in inherently Th2-skewed BALB/c mice. Thus, BcfA functions as both an antigen and an adjuvant, providing protection as a single-component vaccine. BcfA-adjuvanted vaccines may improve the efficacy and durability of vaccines against bordetellae and other pathogens.

Bordetella bronchiseptica Colonization Limits Efficacy, but Not Immunogenicity, of Live-Attenuated Influenza Virus Vaccine and Enhances Pathogenesis After Influenza Challenge.

Intranasally administered live-attenuated influenza virus (LAIV) vaccines provide significant protection against heterologous influenza A virus (IAV) challenge. However, LAIV administration can modify the bacterial microbiota in the upper respiratory tract, including alterations in species that cause pneumonia. We sought to evaluate the effect of Bordetella bronchiseptica colonization on LAIV immunogenicity and efficacy in swine, and the impact of LAIV and IAV challenge on B. bronchiseptica colonization and disease. LAIV immunogenicity was not significantly impacted by B. bronchiseptica colonization, but protective efficacy against heterologous IAV challenge in the upper respiratory tract was impaired. Titers of IAV in the nose and trachea of pigs that received LAIV were significantly reduced when compared to non-vaccinated, challenged controls, regardless of B. bronchiseptica infection. Pneumonia scores were higher in pigs colonized with B. bronchiseptica and challenged with IAV, but this was regardless of LAIV vaccination status. While LAIV vaccination provided significant protection against heterologous IAV challenge, the protection was not sterilizing and IAV replicated in the respiratory tract of all LAIV vaccinated pig. The interaction between IAV, B. bronchiseptica, and host led to development of acute-type B. bronchiseptica lesions in the lung. Thus, the data presented do not negate the efficacy of LAIV vaccination, but instead indicate that controlling B. bronchiseptica colonization in swine could limit the negative interaction between IAV and Bordetella on swine health.

Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4⁺CD25⁺ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice.

The respiratory tract is constantly exposed to the environment and displays a favorable niche for colonizing microorganisms. However, the effects of respiratory bacterial carriage on the immune system and its implications for secondary responses remain largely unclear. We have employed respiratory carriage with Bordetella bronchiseptica as the underlying model to comprehensively address effects on subsequent immune responses. Carriage was associated with the stimulation of Bordetella-specific CD4⁺, CD8⁺, and CD4⁺CD25⁺Foxp3⁺ T cell responses, and broad transcriptional activation was observed in CD4⁺CD25⁺ T cells. Importantly, transfer of leukocytes from carriers to acutely B. bronchiseptica infected mice, resulted in a significantly increased bacterial burden in the recipient's upper respiratory tract. In contrast, we found that respiratory B. bronchiseptica carriage resulted in a significant benefit for the host in systemic infection with Listeria monocytogenes. Adaptive responses to vaccination and influenza A virus infection, were unaffected by B. bronchiseptica carriage. These data showed that there were significant immune modulatory processes triggered by B. bronchiseptica carriage, that differentially affect subsequent immune responses. Therefore, our results demonstrated the complexity of immune regulation induced by respiratory bacterial carriage, which can be beneficial or detrimental to the host, depending on the pathogen and the considered compartment.

Membrane Vesicles Derived from Bordetella bronchiseptica: Active Constituent of a New Vaccine against Infections Caused by This Pathogen.

Bordetella bronchiseptica, a Gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including humans (albeit rarely). We recently designed Bordetella pertussis and Bordetella parapertussis experimental vaccines based on outer membrane vesicles (OMVs) derived from each pathogen, and we obtained protection against the respective infections in mice. Here, we demonstrated that OMVs derived from virulent-phase B. bronchiseptica (OMVBbvir+) protected mice against sublethal infections with different B. bronchiseptica strains, two isolated from farm animals and one isolated from a human patient. In all infections, we observed that the B. bronchiseptica loads were significantly reduced in the lungs of vaccinated animals; the lung-recovered CFU were decreased by ≥4 log units, compared with those detected in the lungs of nonimmunized animals (P < 0.001). In the OMVBbvir+-immunized mice, we detected IgG antibody titers against B. bronchiseptica whole-cell lysates, along with an immune serum having bacterial killing activity that both recognized B. bronchiseptica lipopolysaccharides and polypeptides such as GroEL and outer membrane protein C (OMPc) and demonstrated an essential protective capacity against B. bronchiseptica infection, as detected by passive in vivo transfer experiments. Stimulation of cultured splenocytes from immunized mice with OMVBbvir+ resulted in interleukin 5 (IL-5), gamma interferon (IFN-γ), and IL-17 production, indicating that the vesicles induced mixed Th2, Th1, and Th17 T-cell immune responses. We detected, by adoptive transfer assays, that spleen cells from OMVBbvir+-immunized mice also contributed to the observed protection against B. bronchiseptica infection. OMVs from avirulent-phase B. bronchiseptica and the resulting induced immune sera were also able to protect mice against B. bronchiseptica infection.IMPORTANCEBordetella bronchiseptica, a Gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including humans (albeit rarely). Several vaccines aimed at preventing B. bronchiseptica infection have been developed and used, but a safe effective vaccine is still needed. The significance and relevance of our research lie in the characterization of the OMVs derived from B. bronchiseptica as the source of a new experimental vaccine. We demonstrated here that our formulation based on OMVs derived from virulent-phase B. bronchiseptica (OMVBbvir+) was effective against infections caused by B. bronchiseptica isolates obtained from different hosts (farm animals and a human patient). In vitro and in vivo characterization of humoral and cellular immune responses induced by the OMVBbvir+ vaccine enabled a better understanding of the mechanism of protection necessary to control B. bronchiseptica infection. Here we also demonstrated that OMVs derived from B. bronchiseptica in the avirulent phase and the corresponding induced humoral immune response were able to protect mice from B. bronchiseptica infection. This realization provides the basis for the development of novel vaccines not only against the acute stages of the disease but also against stages of the disease or the infectious cycle in which avirulence factors could play a role.

Comparative efficacy of intranasal and injectable vaccines in stimulating Bordetella bronchiseptica-reactive anamnestic antibody responses in household dogs.

In order to determine the comparative efficacy of injectable and intranasal vaccines to stimulate Bordetella bronchiseptica (Bb)-reactive anamnestic antibodies, a trial was conducted using 144 adult household dogs of various breeds and ages, which had been previously administered intranasal Bb vaccine approximately 12 months before enrollment. Dogs were randomized into 2 groups and blood, nasal swabs, and pharyngeal swabs were collected prior to the administration of single component Bb vaccines intranasally or parenterally. Ten to 14 days later all dogs were resampled to measure changes in systemic and local antibody to Bb. There were no differences in the changes in Bb-reactive serum IgG and nasal IgA between the groups, whereas intranasally vaccinated dogs had significantly higher Bb-reactive serum IgA. These data indicate that both of the current generation of intranasal (modified-live) and injectable (acellular) Bb vaccines can stimulate anamnestic local and systemic antibody responses in previously vaccinated, Bb-seropositive adult household dogs.

Efficacité comparative des vaccins intranasaux et injectables pour stimuler les réponses des anticorps anamnestiques réagissant àBordetella bronchisepticachez les chiens domestiques. Afin de déterminer l'efficacité comparative des vaccins injectables et intranasaux pour stimuler les anticorps anamnestiques réagissant à Bordetella bronchiseptica (Bb), un essai a été réalisé à l'aide de 144 chiens domestiques adultes de diverses races et d'âges différents, auxquels l'on avait déjà administré le vaccin Bb intranasal environ 12 mois avant le recrutement. Les chiens ont été assignés au hasard à deux groupes et des échantillons sanguins, et écouvillons nasaux et pharyngés ont été prélevés avant l'administration de vaccins Bb à composant unique soit par voie intranasale ou parentérale. Dix à 14 jours plus tard, on a prélevé de nouveaux échantillons pour tous les chiens afin de mesurer les changements dans les anticorps systémiques et locaux pour Bb. Il n'y avait aucune différence au niveau des changements pour l'IgG sérique et l'IgA nasal réactif à Bb entre les groupes, tandis que les chiens vaccinés par voie intranasale présentaient un niveau significativement supérieur d'IgA sériques réactives à Bb. Ces données indiquent que les deux générations actuelles de vaccins Bb intranasal (vivant modifié) et injectable (acellulaire) peuvent stimuler les réponses locale et systémique des anticorps Bb chez les chiens adultes domestiques antérieurement vaccinés.(Traduit par Isabelle Vallières).

The Bordetella Bps Polysaccharide Is Required for Biofilm Formation and Enhances Survival in the Lower Respiratory Tract of Swine.

Bordetella bronchiseptica is pervasive in swine populations and plays multiple roles in respiratory disease. Additionally, B. bronchiseptica is capable of establishing long-term or chronic infections in swine. Bacterial biofilms are increasingly recognized as important contributors to chronic bacterial infections. Recently the polysaccharide locus bpsABCD has been demonstrated to serve a critical role in the development of mature biofilms formed by the sequenced laboratory strain of B. bronchiseptica We hypothesized that swine isolates would also have the ability to form mature biofilms and the bpsABCD locus would serve a key role in this process. A mutant containing an in-frame deletion of the bpsABCD structural genes was constructed in a wild-type swine isolate and found to be negative for poly-N-acetylglucosamine (PNAG)-like material by immunoblot assay. Further, the bpsABCD locus was found to be required for the development and maintenance of the three-dimensional structures under continuous-flow conditions. To investigate the contribution of the bpsABCD locus to the pathogenesis of B. bronchiseptica in swine, the KM22Δbps mutant was compared to the wild-type swine isolate for the ability to colonize and cause disease in pigs. The bpsABCD locus was found to not be required for persistence in the upper respiratory tract of swine. Additionally, the bpsABCD locus did not affect the development of anti-Bordetella humoral immunity, did not contribute to disease severity, and did not mediate protection from complement-mediated killing. However, the bpsABCD locus was found to enhance survival in the lower respiratory tract of swine.

Modifications of Bordetella bronchiseptica core lipopolysaccharide influence immune response without affecting protective activity.

Bordetella bronchiseptica produces respiratory disease primarily in mammals including humans. Although a considerably amount of research has been generated regarding lipopolysaccharide (LPS) role during infection and stimulating innate and adaptive immune response, mechanisms involved in LPS synthesis are still unknown. In this context we searched in B. bronchiseptica genome for putative glycosyltransferases. We found possible genes codifying for enzymes involved in sugar substitution of the LPS structure. We decided to analyse BB3394 to BB3400 genes, closed to a previously described LPS biosynthetic locus in B. pertussis. Particularly, conservation of BB3394 in sequenced B. bronchiseptica genomes suggests the importance of this gene for bacteria normal physiology. Deletion of BB3394 abolished resistance to naive serum as described for other LPS mutants. When purified LPS was analyzed, differences in the LPS core structure were found. Particularly, a GalNA branched sugar substitution in the core was absent in the LPS obtained from BB3394 deletion mutant. Absence of GalNA in core LPS alters immune response in vivo but is able to induce protective response against B. bronchiseptica infection.

Bordetella bronchiseptica antigen enhances the production of Mycoplasma hyopneumoniae antigen-specific immunoglobulin G in mice.

We previously demonstrated that Bordetella (B.) bronchiseptica antigen (Ag) showed high immunostimulatory effects on mouse bone marrow cells (BMs) while Mycoplasma (M.) hyopneumoniae Ag showed low effects. The focus of this study was to determine if B. bronchiseptica Ag can enhance the M. hyopneumoniae Ag-specific immune response and whether the host's immune system can recognize both Ags. MTT assay results revealed that each or both Ags did not significantly change BM metabolic activity. Flow cytometry analysis using carboxyfluorescein succinimidyl ester showed that B. bronchiseptica Ag can promote the division of BMs. In cytokine and nitric oxide (NO) assays, B. bronchiseptica Ag boosted production of tumor necrosis factor-alpha in M. hyopneumoniae Ag-treated BMs, and combined treatment with both Ags elevated the level of NO in BMs compared to that from treatment of M. hyopneumoniae Ag alone. Immunoglobulin (Ig)G enzyme-linked immunosorbent assay using the sera of Ag-injected mice clearly indicated that B. bronchiseptica Ag can increase the production of M. hyopneumoniae Ag-specific IgG. This study provided information valuable in the development of M. hyopneumoniae vaccines and showed that B. bronchiseptica Ag can be used both as a vaccine adjuvant and as a vaccine Ag.

Prior exposure to Bordetella species as an exclusion criterion in the baboon model of pertussis.

The baboon model of Bordetella pertussis infection is the newest and most clinically accurate model of the human disease to date. However, among the 15 experimentally infected baboons in this study, a subset of baboons did not exhibit the expected high bacterial colonization levels or increase in white blood cell count. Moreover, cultures of nasopharyngeal wash samples from several baboons suggested B. bronchiseptica coinfection. Analysis of serum antibodies recognizing filamentous hemagglutinin, pertussis toxin and B. pertussis lipo-oligosaccharide indicated that several baboons had likely been previously exposed to Bordetella species and that prior exposure correlated with partial protection from B. pertussis infection. Notably, all animals with a baseline Fha titer of 5 IU/ml or below exhibited symptoms typical of the model, suggesting this value can be used as inclusion criteria for animals prior to study enrollment. While B. pertussis infection is endemic to human populations and B. bronchiseptica is common in wild small mammals, this study illustrates that baboons can readily harbor both organisms. Awareness of Bordetella species that share antigens capable of generating protective immune responses and tracking of prior exposure to those species is required for successful use of the baboon model of pertussis.

Comparison of ribotyping and sequence-based typing for discriminating among isolates of Bordetella bronchiseptica.

PvuII ribotyping and MLST are each highly discriminatory methods for genotyping Bordetella bronchiseptica, but a direct comparison between these approaches has not been undertaken. The goal of this study was to directly compare the discriminatory power of PvuII ribotyping and MLST, using a single set of geographically and genetically diverse strains, and to determine whether subtyping based on repeat region sequences of the pertactin gene (prn) provides additional resolution. One hundred twenty-two isolates were analyzed, representing 11 mammalian or avian hosts, sourced from the United States, Europe, Israel and Australia. Thirty-two ribotype patterns were identified; one isolate could not be typed. In comparison, all isolates were typeable by MLST and a total of 30 sequence types was identified. An analysis based on Simpson's Index of Diversity (SID) revealed that ribotyping and MLST are nearly equally discriminatory, with SIDs of 0.920 for ribotyping and 0.919 for MLST. Nonetheless, for ten ribotypes and eight MLST sequence types, the alternative method discriminates among isolates that otherwise type identically. Pairing prn repeat region typing with ribotyping yielded 54 genotypes and increased the SID to 0.954. Repeat region typing combined with MLST resulted in 47 genotypes and an SID of 0.944. Given the technical and practical advantages of MLST over ribotyping, and the nominal difference in their SIDs, we conclude MLST is the preferred primary typing tool. We recommend the combination of MLST and prn repeat region typing as a high-resolution, objective and standardized approach valuable for investigating the population structure and epidemiology of B. bronchiseptica.

Comparative efficacy of intranasal and oral vaccines against Bordetella bronchiseptica in dogs.

In order to determine the comparative efficacy of vaccines administered intranasally or orally to protect puppies from disease subsequent to experimental infection with Bordetella bronchiseptica (Bb), a randomized controlled trial was performed using 48 approximately 8-week-old specific pathogen free, Bb naive Beagle puppies. Puppies were randomized into three groups and administered vaccines containing Bb intranasally or orally, or a placebo intranasally. Twenty-one days later, all dogs were challenge exposed via aerosol administration of Bb. Clinical signs, nasal bacterial shedding and immune responses were monitored for 28 days after challenge. Intranasally vaccinated puppies had significantly lower rates of coughing, nasal discharge, retching and sneezing (i.e. were less sick clinically) than control puppies. The distinction between the orally vaccinated puppies and the control puppies was less consistent. The orally vaccinated puppies had less coughing and less retching than the control puppies, but nasal discharge and sneezing did not differ from control animals. Orally vaccinated puppies had higher rates of coughing, nasal discharge, retching and sneezing than the intranasally vaccinated puppies. Although both intranasal and oral Bb vaccines stimulated immune responses associated with disease sparing following Bb infection, the intranasal route of delivery conferred superior clinical outcomes. The observed difference in clinical efficacy suggests the need to question the rationale for the use of currently available orally administered Bb vaccines.

Heterologous expression of porcine reproductive and respiratory syndrome virus glycoprotein 5 in Bordetella bronchisepticaaroA mutant.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important disease around the globe. Protection against this virus remains problematic. Here, we evaluated antibody (IgG & IgA) inducibility of a heterologous PRRSV glycoprotein 5 (GP5) expressed in a live attenuated Bordetella bronchisepticaaroA mutant strain (BBS-GP5). Mice and pigs were primed with recombinant GP5 (rGP5) subcutaneously followed by boosting with live BBS-GP5. As a result, anti-GP5 IgG was induced in both mice (P<0.001) and pigs (P<0.1). Pigs were challenged with live PRRSV (VR2332). Viral RNA was found to be significantly (P<0.01) removed in the vaccinated pig group. Overall, BBS-GP5 is a good candidate as a live attenuated vaccine against PRRSV infection.